Luminescent nucleic acid stains are molecules that non-covalently associate with oligonucleotides or nucleic acids, and are either intrinsically luminescent (typically fluorescent) or that display a change in their spectral characteristics upon associating with ox nucleic acids. The rapid development of new fluorescent nucleic acid stains has resulted in the use of fluorescence-based methods in a wide variety of studies directed at or using nucleic acids.
Typically the addition of a nucleic acid stain results in the fluorescent staining of most or all nucleic acid fragments present in the sample. The resulting undifferentiated staining often limits the utility of nucleic acid stains in some assays, or necessitates additional separation steps either before or after staining.
However, when some of the nucleic acid fragments (e.g. oligonucleotides) are covalently labeled with a quenching moiety, the fluorescence intensity of the associated nucleic acid stain molecules in the vicinity is decreased or even fully quenched. The quenching moiety may emit fluorescence at a wavelength longer than that of the nucleic acid stain, it may be dimly fluorescent, or essentially nonfluorescent.
Energy transfer pairs where both the donor and acceptor are covalently bound to the same nucleic acid are known. Such energy transfer pairs have been used to detect changes in oligonucleotide conformation, such as in Tyagi et al. (EP 0 745 690 A2 (1996)) and Pitner et al. (U.S. Pat. No. 5,691,145 (1997)). They also have been used to detect cleavage of the oligonucleotide at a point between the donor and acceptor dyes, such as in Han et al. (U.S. Pat. No. 5,763,181 (1998)), Nadeau et al. (U.S. Pat. No. 5,846,726 (1998)), and Wang et al. (ANTIVIRAL CHEMISTRY & CHEMOTHERAPY 8, 303 (1997)). Energy transfer pairs covalently bound to oligonucleotides have also been used to provide a shift in the ultimate emission wavelength upon excitation of the donor dye, such as by Ju (U.S. Pat. No. 5,804,386 (1998)).
The combination of a non-covalently bound nucleic acid stain with a covalently attached fluorophore on a single-stranded oligonucleotide hybridization probe has been used to detect specific DNA target sequences by monitoring the fluorescence of either the nucleic acid stain or the covalent label, such as in Lee and Fuerst (PCT Int. Appl. WO 99 28,500).
However, the use of a covalently bound acceptor moiety to decrease the fluorescence of noncovalently associated fluorescent nucleic acid stains has not previously been described. In addition, the use of an essentially nonfluorescent, covalently bound acceptor dye for the purpose of quenching the fluorescence of multiple fluorescent nucleic acid stain molecules bound at the same time to the same oligonucleotide has not been previously described, particularly where the oligonucleotide is a primer for a nucleic acid amplification or elongation reaction.
The methods of the present invention permit a significant reduction in background fluorescence levels where fluorescent nucleic acid stains are used in assays that require the presence of large numbers of oligonucleotide primers. In particular, the contribution to total fluorescence due to nucleic acid stains associated with primers, or primer dimers, can be substantially reduced or essentially eliminated. The reagents and methods described herein also permit continuous assays of chain elongation that do not require reagent addition or separation steps.